DNA条形码与FTIR在石斛分析中的联用

目前市面上石斛种类混淆和以次充好现象的频发。为建立石斛快速分析鉴定体系,测试常用的植物DNA条形码能否适用于石斛鉴定,并探讨DNA条形码与FTIR联用的可行性,本研究选择常用的植物DNA条形码并结合FTIR技术对市面上常见的9种石斛进行了分析。结果表明,9种石斛的ITS2序列的种间遗传距离最小值大于种内遗传距离最大值,且平均种间遗传距离为平均种内遗传距离的58倍。ITS2序列具有作为石斛鉴定DNA条形码的潜能,且优于psb A-trn H序列的石斛鉴别能力。此外,基于FTIR还获取了9种石斛的特异性红外光谱,并基于峰形、峰高、峰强等进行了简单的组分分析,发现金钗石斛、铁皮石斛、铜皮石斛和密花石斛具有相对较高的含糖量。本研究首次将DNA条形码与FTIR技术联用,为石斛的快速分析鉴定提供了可能。     

DNA条形码与实时荧光定量PCR技术在铁皮石斛鉴定中的应用

为建立铁皮石斛准确、高效的鉴定体系,本研究以101份石斛属和蝴蝶兰属植物样品为试验材料,通过对比ITS、psbA-trnH、matK及rbcL基因在石斛属植物中的鉴定能力,筛选出ITS作为本研究最理想的DNA条形码。以ITS序列作为靶基因,设计铁皮石斛特异引物和特异探针,以及石斛属通用引物和探针,利用实时荧光定量PCR(TaqMan)技术,建立铁皮石斛多重实时荧光PCR检测新体系,通过特异性、灵敏度和实际样本验证,发现参试样品中的25份铁皮石斛均可被有效鉴定,与其他鉴定方法相比,该方法具有特异性强、灵敏度高(高出普通PCR 100倍)、重复性好且高效经济的优势。本研究结果对铁皮石斛的资源保护与利用起到了积极作用。     

铁皮石斛生物技术研究进展

铁皮石斛是一种名贵中药材。近年来,因为需求量的增加和自然繁殖困难等原因,野生资源日趋濒危。除原生地保护之外,利用生物技术进行人工繁殖和种质改良是解决这一问题的有效途径。本文就近年来有关铁皮石斛生物技术方面的研究进展进行简要综述,重点对种质资源的离体保存、遗传多样性和鉴定、组织培养、诱变育种和基因工程技术等方面的研究进行了综述,同时对铁皮石斛今后的开发利用工作提出了建议。     

铁皮石斛EST-SSR分子标记的开发

为了开发铁皮石斛EST-SSR分子标记,利用SSRIT软件对铁皮石斛近缘物种-金钗石斛的15 383条EST序列进行SSR位点查找,利用Primer 5和Oligo 7软件进行引物设计和评价,之后经PCR扩增和琼脂糖凝胶电泳对引物进行初步筛选,并通过聚丙烯酰氨凝胶电泳对所筛选的引物进行有效性检测。结果表明,SSRIT软件共查找到370条SSR位点序列,共有379个SSR位点,候选SSR位点出现的频率为2.46%。二核苷酸、三核苷酸和六核苷酸重复是最主要的重复类型,分别占41.42%、26.91%和27.44%,其中二核苷酸重复中AG/CT(20.84%)和GA/TC(18.73%)最丰富;三核苷酸重复中TCT/AGA(4.22%)和GA/TC(3.96%)最丰富;而六核苷酸重复基元较多。EST-SSR平均长度为24 bp,平均每8.5条EST序列包含1个SSR位点。设计的106对EST-SSR引物中有50对能扩增出理想的PCR产物,引物有效扩增率为47.17%,其中有43对能够扩增出多态性条带,占可扩增引物的86%。本研究开发的43对引物为铁皮石斛遗传多样性分析、种质鉴定、遗传图谱构建和功能基因研究等提供了重要的参考价值。     

石首鱼科海洋鱼类DNA条形码的构建

为筛选石首科鱼类物种鉴定的最优DNA条形码,本试验对大黄鱼、小黄鱼、黄姑鱼和棘头梅童鱼4种鱼类71份样本的细胞色素氧化酶亚基I(COI)、线粒体控制区(D-loop)和16S rRNA 3种基因序列进行PCR扩增和测序,分析比较各基因序列比对鉴定能力、种内和种间差异、barcoding gap检验以及序列多态性。结果表明,3种候选DNA条形码序列的种间遗传距离均高于种内遗传距离,符合作为DNA条形码的基本要求。通过遗传差异和barcoding gap检验分析发现,COI和D-loop区适合作为鉴定大黄鱼、小黄鱼、黄姑鱼和棘头梅童鱼4种石首科鱼类的DNA条形码;16S rRNA基因存在一定的缺陷,但是D-loop区和16S rRNA基因序列可能存在比COI通用序列更为有效的DNA条形码区域。考虑引物通用性,推荐COI基因作为最适DNA条形码。本研究结果为海洋鱼类的快速鉴定提供了一定的参考依据。     

铁皮石斛SCoT-PCR反应体系构建及优化

采用正交设计和单因素试验相结合的方法,对铁皮石斛的SCoT-PCR反应中5个因素(DNA 模板、Mg2+、dNTPs、Taq 酶和引物)进行优化试验。建立了适合铁皮石斛既稳定且多态性高的SCoT-PCR的最佳反应体:20μl PCR反应体系中含有1×Buffer、30ng DNA 模板、2.5mmol·L-1Mg2+、0.15mmol·L-1 dNTPs、1.5U Taq DNA聚合酶和0.4μmol·L-1引物。对铁皮石斛的SCoT-PCR最佳反应体系的退火温度进行梯度试验,发现本试验引物S6的退火温度为54.5℃,且最适退火温度因引物而异。这一优化的SCoT-PCR反应体系在32份铁皮石斛材料的验证中表现出良好的稳定性和重复性。该优化的SCoT-PCR反应体系为进一步进行铁皮石斛种质鉴定、遗传图谱构建、基因定位和遗传多样性的分析奠定了技术基础。     

我国南方野生樱属植物的分子系统发育分析

为鉴定我国樱属植物的系统发育关系,本研究以30种典型樱属植物为试验材料,利用核基因ITS和4个叶绿体间隔序列[petA-psbJ、trnH-psbA、rpl32-trnL(UAG)和trnL-trnF]进行测序,采用MEGA 6软件对5个序列特点进行分析,并构建系统发育树。结果表明,5个序列的DNA片段长度多态性和单核苷酸多态性较高,其中ITS和rpl32-trnL(UAG)变异程度最大,可作为樱属植物核心条形码序列,其他序列可作为辅助序列;通过5个序列的组合序列构建的系统发育树,可相对准确地反映樱属植物间的系统发育关系。本研究结果为进一步探究樱属植物的起源、系统发育关系、物种鉴定及资源保护提供了一定的参考依据。     

基于EST-SSR标记的MCID法鉴定冬瓜种质资源

种质资源鉴定是研究与利用冬瓜种质资源的基础。为了明确冬瓜种质资源遗传多样性,本研究基于表达序列标签-简单重复序列(EST-SSR)分子标记的人工绘制品种鉴别示意图(MCID)法鉴定40份冬瓜种质资源。利用筛选出的7对引物对冬瓜种质资源进行PCR扩增,应用MCID法构建冬瓜品种鉴定图。结果表明,利用7对引物扩增的特征性谱带能够将冬瓜品种在分子水平上区分开,同时能够构建40份冬瓜种质资源的品种鉴定图。利用Ntsys2.10e软件进行聚类分析,在遗传相似系数0.77处,将冬瓜资源分为3个类群。品种鉴定图能够快速地找到区分品种的引物,比聚类图更加直观。基于EST-SSR标记的MCID法是一种有效的冬瓜资源鉴定方法。本研究对冬瓜资源鉴定具有重要意义。     

基于SNP标记的504份番茄种质资源遗传多样性分析

为深入了解番茄种质资源的遗传多样性,运用竞争性等位基因特异性PCR(KASP)技术,利用前期筛选出的60对多态性较高的单核苷酸多态性标记(SNP),对收集的504份番茄种质资源进行遗传多样性分析、聚类分析、主成分分析及群体结构分析。结果表明,60个SNP分子标记共检测到181个等位基因,基因多样性平均值为0.450,期望杂合率(He)平均值为0.069,多态性信息值(PIC)变化范围为0.171~0.583,平均值为0.381。在遗传距离为0.36时,504份番茄材料被划分为7个类群,各材料间的平均遗传距离为0.62;根据主成分分析结果将群体分为3个类群;基于SNP标记对参试材料进行群体结构分析,在K=3时,504份番茄种质资源被划分为3类。本研究筛选出的60对SNP标记的多态性为中度偏高,番茄种质资源遗传多样性较丰富,可为后续宁夏地区番茄的核心种质构建及种质资源的有效利用提供理论依据。     

三叶青突变体的理化成分评价与分子鉴定

为探究三叶青品种泽青1号是否产生药理成分突变,对26份三叶青样本的可溶性多糖、总酮、总皂苷、总酚、蛋白质、矿物质及游离氨基酸进行了理化评价与相关性分析,并检测了其ITS序列的位点突变情况。结果筛选出103、107、109、112、113、125、126、127、130、131、132等优异突变体。泽青1号突变体类群与其23个近缘崖爬藤属种间同源性为49%、平均遗传距离为0.54、组间距离为0.993;泽青1号突变体间同源性为93%~99%、配对距离为0.006 40~0.116 96、组内距离为0.060 70,成功构建了基于ITS种属鉴定的突变体DNA条形码。本研究初步筛选出11个具有多元化定向育种应用价值的突变体,为解决三叶青种质资源稀缺、退化的问题提供了材料基础;同时基于ITS分子标记成功鉴定并区分泽青1号突变体类群与其他近缘崖爬藤属物种,筛选出突变类群的突变位点,为三叶青突变体种质资源评价提供了技术支撑。     

低氮和干旱胁迫对富士和秦冠生长及氮素利用的影响

【目的】以富士（Fuji）、 秦冠（Qinguan）嫁接在平邑甜茶（Malus hupehensis Rehd.）上的当年生盆栽苗为试验材料,采用砂培方法,研究了缺氮胁迫和干旱对富士和秦冠生长情况、 光合参数、 植株各部位氮磷钾含量及氮素利用效率的影响,分析比较了低氮干旱条件下富士和秦冠生长及氮素利用的差异,以期为果树生产高效肥水利用提供理论指导。【方法】试验共设四个处理: 正常氮正常水（ZZ）、 低氮正常水（DZ）、 正常氮干旱（ZG）、 低氮干旱（DG）。氮素和水分均设置两个水平,分别为正常氮（6 mmol/L NO-3-N）、 低氮（0.3 mmol/LNO-3-N）、 正常供水（保持盆中砂子相对含水量为饱和含水量的80%~85%）、 干旱处理（保持盆中砂子相对含水量为饱和含水量的60%~65%）。【结果】富士和秦冠的生物量（茎和叶）、 株高茎粗等生长指标以及光合速率、 气孔导度、 蒸腾速率均为正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）,并且相对应处理下秦冠的以上指标均高于富士;正常供水下,缺氮处理使富士、 秦冠的根冠比比正常氮处理均有所增加,富士提高了2.05%,秦冠提高了22.40%。富士和秦冠的氮、 磷、 钾含量均表现出正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）; 氮、 钾元素含量在植株各部位的分布顺序依次是叶＞根＞茎,磷元素则是根＞叶＞茎;光合氮素利用效率（PNUE）和氮素利用效率表现为秦冠处理之间差异极显著,富士处理之间差异不显著;秦冠的PNUE和NUE明显高于富士,在低氮正常水（DZ）处理下,秦冠氮肥利用率比富士高42.07%,在低氮干旱（DG）处理下高64.14%;低氮胁迫下富士和秦冠的NUE显著提高,并且秦冠提高的幅度高于富士。【结论】施用氮肥能够显著提高富士与秦冠的干物质量,同等水肥条件下,秦冠生长优于富士;水分亏缺会减少叶片对氮的吸收,干旱条件下适度增施氮肥,可提高果树的抗旱能力;低氮干旱胁迫下秦冠的生长指标、 光合指标及氮素利用效率指标均优于富士,表现出较强的抗低氮干旱胁迫的能力。     

Pasture Vegetation Elemental Analysis by Laser-Induced Breakdown Spectroscopy

Laser-induced breakdown spectroscopy (LIBS) is a new technique for the analysis of plant material. This study investigates the application of LIBS to pasture-based plant samples. The LIBS measurements were obtained from pelletized pasture samples (100 samples) that had been also analyzed by inductively coupled plasma–optical emission spectroscopy (ICP-OES) following microwave digestion for calibration and comparison purposes. Comparisons for elements sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), boron (B), phosphorus (P), and sulfur (S) showed that LIBS could be used for almost all the standard profile total elements with concentrations down to low mg/kg levels (observed error of Na: 0.024 percent, K: 0.18 percent, Mg: 0.016 percent, Ca: 0.073 percent, P: 0.017 percent, Mn: 31 mg/kg, Fe: 150 mg/kg, Zn: 6.6 mg/kg, and B: 1.1 mg/kg). Elemental analysis at less than mg/kg levels was not possible using LIBS. The elements S and Cu were particularly difficult to analyze with reliability using LIBS at the concentration levels found in the plant samples. Replacing microwave digestion and subsequent ICP analysis with a direct analysis of dried plant samples using LIBS has the potential to improve the productivity and reduce the cost of testing.     

Faculty positions:Center for Agricultural Resources Research,Chinese Academy of Sciences

正The Center for Agricultural Resources Research(CARR),the Institute of Genetics and Developmental Biology(IGDB),Chinese Academy of Sciences,invites applicants for several research group leader positions.CARR is one of the research organizations in Chinese Academy of Sciences(CAS).We seek nominations and applications from individuals who have expertise and a record of accomplishment in research areas related to ecology,agro-hydrology,     

Macroinvertebrates in North American tallgrass prairie soils: effects of fire, mowing, and fertilization on density and biomass

The responses of tallgrass prairie plant communities and ecosystem processes to fire and grazing are well characterized. However, responses of invertebrate consumer groups, and particularly soil-dwelling organisms, to these disturbances are not well known. At Konza Prairie Biological Station, we sampled soil macroinvertebrates in 1994 and 1999 as part of a long-term experiment designed to examine the effects and interactions of annual fire, mowing, and fertilization (N and P) on prairie soil communities and processes. For nearly all taxa, in both years, responses were characterized by significant treatment interactions, but some general patterns were evident. Introduced European earthworms (Aporrectodea spp. and Octolasion spp.) were most abundant in plots where fire was excluded, and the proportion of the total earthworm community consisting of introduced earthworms was greater in unburned, unmowed, and fertilized plots. Nymphs of two Cicada genera were collected (Cicadetta spp. and Tibicen spp.). Cicadetta nymphs were more abundant in burned plots, but mowing reduced their abundance. Tibicen nymphs were collected almost exclusively from unburned plots. Treatment effects on herbivorous beetle larvae (Scarabaeidae, Elateridae, and Curculionidae) were variable, but nutrient additions (N or P) usually resulted in greater densities, whereas mowing usually resulted in lower densities. Our results suggest that departures from historical disturbance regimes (i.e. frequent fire and grazing) may render soils more susceptible to increased numbers of European earthworms, and that interactions between fire, aboveground biomass removal, and vegetation responses affect the structure and composition of invertebrate communities in tallgrass prairie soils.     

Variation in quality parameters between and within 14 Nordic tree fruit and berry species

Abstract

A 3-year study was carried out to investigate quality parameters in 14 tree fruit and berry species grown in southern Norway. The species were blueberry, apple, aronia, sour cherry, sweet cherry, red raspberry, strawberry, blackcurrant, gooseberry, red currant and elderberry, harvested along with wild bilberry, cloudberry and lingonberry. Significant differences between species were identified for all quality parameters. The coefficient of variation between species was lowest for pH (12.5%), dry matter (18.9%) and soluble solids (25.3%), followed by titratable acids (59.3%), total phenolics (83.8%), antioxidant capacity FRAP (85.7%) and antiradical power by the DPPH-assay (97.8%), total monomeric anthocyanins (132%) and ascorbic acid (137%). Average coefficient of variation within species were lower and ranged from 4 (pH) to 62% (ascorbic acid). Only the FRAP values were significantly affected by harvesting year with lower levels in 2004 than in 2005 and 2006. There were significant interactions between species and harvesting year for dry matter, soluble solids, pH, ascorbic acid and FRAP. The results indicate generic ranges in composition within species independent upon growing location and climate, and the composition of the tree fruits and berries is not likely to deviate from these ranges. It is concluded that desirable composition of tree fruits and berries and their products should primarily be achieved by selection among species rather than searching fors broadened variation within individual species.     

Potassium Fixation and Release Characteristics in Rhizosphere and Nonrhizosphere Soils for a Rapeseed–Rice Cropping Sequence

Potassium (K) fixation and release in soil are important factors in the long-term sustainability of a cropping system. Changes in K concentration and characteristics of K fixation and release in rhizosphere and nonrhizosphere soils in the rapeseed (Brassica napus L.)–rice (Oryza sativa L.) rotation were investigated using a rhizobox system. The concentrations of different forms of K in both rhizosphere and nonrhizosphere soils decreased with plants compared to without plants, regardless of K fertilizer application. Potassium uptake by crops mainly came from the rhizosphere soil. In the treatment without K fertilizer (–K), the main form of K supplied by the soil to the crops was 1.0 mol L^?1 nitric acid (HNO₃) nonextractable K, followed by nonexchangeable K, and then exchangeable K. In the treatment with K fertilizer (+K), the main K forms supplied by the soil to the crops were exchangeable K and nonexchangeable K. The amount and rate of K fixation after one cycle of the rapeseed–rice rotation was greater in rhizosphere soil than in nonrhizosphere soil. The amount and rate of K fixation of soil in the +K treatment were significantly less than in the –K treatment. The cumulative amounts of K released with 1.0 mol L^?1 ammonium acetate (NH₄OAc) and 1.0 mol L^?1 HNO₃ extraction increased with the increasing numbers of extractions, but the K-releasing power of soil by successive extraction decreased gradually and finally became almost constant. The release of K was less in rhizosphere soil than in nonrhizosphere soil. The release of K in the +K treatment was similar to that in the –K treatment in rhizosphere soil, but the K release in nonrhizosphere soil was greater with the +K than the –K treatment. Overall, the information obtained in this study will be helpful in formulating more precise K fertilizer recommendations for certain soils.     

Electron donor and pH relationships for biologically enhanced dissolution of chlorinated solvent DNAPL in groundwater

Biologically enhanced dissolution offers a method to speed removal of chlorinated solvent dense non-aqueous-phase liquid (DNAPL) sources such as tetrachloroethene (PCE) and trichoroethene (TCE) from aquifers. Bioremediation is accomplished by adding an electron donor to the source zone where fermentation to intermediates leading to acetic acid and hydrogen results. The hydrogen and possibly acetic acid are used by dehalogenating bacteria to convert PCE and TCE to ethene and hydrochloric acid. Reductive dehalogenation is thus an acid forming process, and sufficient alkalinity must be present to maintain a near neutral pH. The bicarbonate alkalinity required to maintain pH above 6.5 is a function of the electron donor: 800 mg/L of bicarbonate alkalinity is sufficient to achieve about 1.2 mM TCE dechlorination with glucose, 1.7 mM with lactate, and a much higher 3.3 mM with formate. Laboratory studies indicate that in mixed culture, formate can be used as an electron donor for complete conversion to ethene, contrary to pure cultures studies indicating it cannot. Various strategies can be used to add electron donor to an aquifer for DNAPL dehalogenation while minimizing pH problems and excessive electron donor usage, including use of injection-extraction wells, dual recirculation wells, and nested injection-extraction wells.     

Biodiversity and ecogeography of wild Lactuca spp. in some European countries

Staff members of the Department of Botany of Palacký University in Olomouc and Gene Bank Department – Workplace Olomouc, Research Institute of Crop Production in Prague, Czech Republic, conducted an expedition in seven European countries (Austria, Czech Republic, France, Germany, Italy, the Netherlands, Switzerland) in August/September 1999 to collect wild Lactuca spp. germplasm and study its geographic distribution, ecology and biodiversity. During the mission, more than 600 locations were visited resulting in the collection of 602 seed samples (accessions) of wild Lactuca species and 13 seed samples of related genera (Chondrilla and Mycelis). Lactuca serriola f. serriola, L. serriola f. integrifolia, L. saligna and L. viminea subsp. chondrilliflora were prevalent in southern Europe (Italy, France), however, only L. serriola was common in central and western Europe (Austria, Czech Republic, Germany, Netherlands, Switzerland). The greatest diversity of Lactuca species was found in France, where also the most seed samples (165) were collected. The most characteristic habitats with a high density of Lactuca spp. populations were observed along roads and highways, grassy ditches, ruderal communities, and dust-heaps. Natural infections by powdery mildew (Erysiphe cichoracearum) and downy mildew (Bremia lactucae) on some wild Lactuca spp. were observed. Recent observations concerning the geographic distribution, population structure, habitats, and natural occurrence of diseases of Lactuca spp. are discussed. This assemblage of genetic resources of Lactuca spp. can serve as the basis of future studies of species diversification, spatial population structure, plant microevolution, domestication processes, and genetic variability of host-parasite interactions.     

Responses of native legume desert trees used for reforestation in the Sonoran Desert to plant growth-promoting microorganisms in screen house

Three slow-growing legume trees used for desert reforestation and urban gardening in the Sonoran Desert of Northwestern Mexico and the Southwestern USA were evaluated whether their growth can be promoted by inoculation with plant growth-promoting bacteria (Azospirillum brasilense and Bacillus pumilus), unidentified arbuscular mycorrhizal (AM) fungi (mainly Glomus sp.), and supplementation with common compost under regular screenhouse cultivation common to these trees in nurseries. Mesquite amargo (Prosopis articulata) and yellow palo verde (Parkinsonia microphylla) had different positive responses to several of the parameters tested while blue palo verde (Parkinsonia florida) did not respond. Survival of all tree species was over 80% and survival of mesquite was almost 100% after 10 months of cultivation. Inoculation with growth-promoting microorganisms induced significant effects on the leaf gas exchange of these trees, measured as transpiration and diffusive resistance, when these trees were cultivated without water restrictions.     

Increase in auxiliary photoprotective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense

Inoculation of wheat seedlings with the plant growth-promoting bacterium Azospirillum brasilense Cd was immobilized in alginate microbeads and, without applying any stress, significantly increased the quantity of several photosynthetic pigments, such as chlorophyll a, chlorophyll b, and the auxiliary photoprotective pigments violaxanthin, zeaxanthin, antheroxanthin, lutein, neoxanthin, and β-carotene. This resulted in greener plants with no apparent visible stress. After monitoring the quantity of photosynthetic pigments for 4 weeks, we observed that inoculated plants had higher quantities of pigments in shoot and stem. The greatest difference in the quantity of all pigments between inoculated and noninoculated plants occurred in the first week of growth. Regardless of treatment, the quantity of pigments in stems was three to four times less than the quantity of these pigments in shoots. Application of Azospirillum, either as liquid inoculant or as alginate microbeads, did not alter the positive effect of the bacteria on pigment production or the positive response of the plants towards A. brasilense Cd inoculation.